In the field of clinical analysis, a method utilizing coupling enzymes of glucokinase (GK) and glucose-6-phosphate dehydrogenase (G6PDH), i.e., a so-called GK/G6PDH coupling enzyme system, has heretofore been employed in determination of glucose or CPK.
This method is based on the following principle: ##STR1##
The symbols used in the above formulae are defined as follows:
CP: creatine phosphate PA0 C: creatine PA0 ADP: adenosine-5'-diphosphate PA0 ATP: adenosine-5'-triphosphate PA0 G6P: glucose-6-phosphate PA0 NAD(P): oxidized form nicotinamide adenine dinucleotide (phosphate) PA0 NAD(P)H: reduced form nicotinamide adenine dinucleotide (phosphate) PA0 6-PGA: 6-phosphogluconate
The above-described reactions (1), (2) and (3) are catalyzed by CPK, GK and G6PDH, respectively. Thus, the CPK measurement follows the equations (1), (2) and (3), and the lucose measurement, the equations (2) and (3).
Conventional glucose or CPK-measuring compositions utilizing the HK/G6PDH coupling enzyme system are of low stability when stored at room temperature in the form of solution and their service lives as reagents at room temperature (18.degree.-35.degree. C.) after dissolving are very short, as described in Methods in Enzymatic Analysis, 2nd English Edition, ed. by Hans Ulrich Bergmeyer (published by Verlag Chemie International), pages 789-793 and 1196-1205 (1974). Thus, an improved measuring composition using heat stable GK has been developed, as described in Japanese Patent Application (OPI) No. 169598/81 (corresponding to U.S. patent application Ser. No. 267,245, now U.S. Pat. No. 4,438,199, and also to EPC Patent Publication No. 43181) (the term "OPI" as used herein means a "published unexamined Japanese patent application").
The above-proposed composition is improved in storage stability at room temperature compared with conventional compositions. Its stability, however, is not sufficiently satisfactory because of instability of coupling enzyme and SH-containing compound contained in the composition. Furthermore, to keep the stability over long periods of time, it is necessary to add a relatively large amount of enzyme. Use of such a large amount of enzyme does not cause any serious problem. However, as the amount of enzyme to be used is decreased, the problem of a drop in rate of reaction and, in its turn, an increase in the time required for the measurement arises. Although this problem is not noticeable in measuring the individual activity of GK and G6PDH, it becomes seriously noticeable when the composition is used in determination of glucose or CPK. This is one of the causes preventing practical use of the composition.
Addition of potassium chloride (KCZ) in the assay mixture or purification medium of GK is described in Methods in Enzymology, published by Academic Press Inc., Vol. 9, pages 381-388 (1966). It is also described that although KCl possesses an action to stabilize GK, while not exerting influences on the rate of reaction (Vm).